Characterisation and potential impacts of Clostridium spp. in agroecosystems

Abstract

This study investigated the behaviour of Clostridium species, with a focus on Clostridium perfringens, in agroecosystems, examining their genetic features, adaptability under environmental pressures, and potential dissemination through agricultural practices. Using a next-generation technologies, whole-genome sequencing of surface water-derived C. perfringens isolates revealed 11 antibiotic resistance genes (ARGs) and 35 virulence factors, with 79% associated with C. perfringens-specific pathogenic traits. This highlights their adaptability and potential risks to human and environmental health. Comparative analyses demonstrated strong genetic similarity to clinical isolates, emphasising the role of agroecosystems as reservoirs and amplifiers of antibiotic resistance and virulence traits. Transcriptome analysis using RNA-Seq revealed 166 significantly differentially expressed genes mapped to 26 KEGG pathways, including 73 up-regulated and 93 down-regulated genes under sublethal antibiotic exposure. Several resistance and virulence-associated genes were up-regulated, enhancing survival and adaptability under stress conditions. Notably, exposure to cefoxitin resulted in nine differentially expressed ARGs, the highest among the tested antibiotic. Concurrently, genes involved in energy-intensive metabolic pathways, such as lipid and carbohydrate metabolism, were notably down-regulated, indicating a shift in bacterial physiology toward a survival-oriented state. These findings emphasise the ecological risks posed by residual antibiotics in agricultural environments, which foster the persistence and pathogenicity of resistant bacterial populations. The study also explored the survival and transfer of C. sporogenes, a surrogate for pathogenic Clostridium species, in irrigation systems. Contaminated irrigation water facilitated the persistence of bacteria in both soil and the phyllosphere of lettuce, with surface irrigation primarily contaminating the soil and spray irrigation targeting the plant phyllosphere. Surface irrigation led to a peak concentration of 5.59 log copy numbers/g in non-rhizosphere soil at day 22, while spray irrigation resulted in an initial phyllosphere concentration of 9.09 log copy numbers/g leaves, which declined to 0.019 by day 42. Although bacterial concentrations decreased over time, trace amounts remained detectable, presenting potential food safety risks. This research highlights the interconnectedness of environmental reservoirs, antibiotic resistance, and agricultural practices. It also highlights the need for integrated management strategies, including enhanced wastewater treatment, rigorous water quality monitoring, and the adoption of sustainable irrigation practices, to mitigate the spread of antibiotic-resistant and pathogenic bacteria in agroecosystems and ensure food safety.